Gpib-lipid bond construct and use thereof

ABSTRACT

The present invention provides a conjugate wherein GP (glycoprotein) Ib and a lipid are bonded via polyalkylene oxide. The present invention also provides a complex (GPIb lipid complex) containing this conjugate and a free lipid. The GPIb lipid complex is expected to have a potential for practical application in a wide range, as a platelet substitute, a pharmaceutical agent for the prophylaxis or treatment of angiopathy, vascular damage and thrombosis, a diagnostic for vWF deficiency and the like, a biological or medical reagent, a reagent for screening platelet aggregation suppressant or antithrombosis, and the like. The GPIb lipid complex of the present invention is also useful as a diagnostic for finding the location of vascular lesion or thrombus formation, or a therapeutic agent thereof. Moreover, the GPIb lipid complex of the present invention is also superior in retention property in blood, which enables continuous expression of a pharmacological action. The conjugate of the present invention is highly utilizable as an active ingredient of the GPIb lipid complex of the present invention.

TECHNICAL FIELD

[0001] The present invention relates to a conjugate of a GPIb and alipid bonded via polyalkylene oxide (hereinafter to be also referred toas a GPIb-lipid conjugate), a lipid complex containing the conjugate anda free lipid, and use thereof.

BACKGROUND ART

[0002] Various glycoproteins (hereinafter to be referred to as GP) arepresent on the surface of a platelet membrane, and are involved in theexpression of platelet functions. Of such platelet membraneglycoproteins, GPIb, GPIIb, GPIIIa, GPIIIb, GPIV, GPIX and the like areknown. Of these, GPIb functions as a receptor of the von WillebrandFactor (hereinafter to be referred to as vWF). GPIb is a heterodimerhaving a molecular weight of 160,000, wherein α chain and β chain arebonded via a disulfide bond (Proc. Natl. Acad. Sci. USA, Vol. 84, pp.5615-5619, 1987, Hematology & Oncology, Vol. 31, No. 1, pp. 5-10, 1995and the like).

[0003] When vascular damages are caused, platelets quickly adhere to thevascular lesion and form a platelet thrombus by aggregation and thelike. In forming the platelet thrombus, vWF plays an important role asan adhesive protein. It is considered that GPIb binds with vWF as itsreceptor and activates or promotes adhesion and aggregation of plateletsvia vWF at said vascular lesion. In addition, the binding of vWF andGPIb functions to stop bleeding at the vascular lesion but also forms apathologic thrombus. Thus, effective use of GPIb for the examination anddiagnosis of vascular lesion, detection of pathologic thrombus, andtreatment thereof is expected.

[0004] Nevertheless, the use of isolated GPIb has not proved successfulin artificial expression of the physiological activity as mentionedabove. In other words, some idea in the aspect of formulation ofpharmaceutical preparations is needed to practically use GPIb as apharmaceutical agent or reagent.

[0005] The present inventors first found earlier that the physiologicalactivity of GPIb could be expressed by preparing a lipid complexcontaining a conjugate of GPIb and a particular lipid, and a generallipid (WO97/29128, JP-A-9-208599, U.S. Pat. No. 6177059, EP-894807-A).

DISCLOSURE OF THE INVENTION

[0006] In view of the above-mentioned situation, the present inventorshave further studied, and as a result, found that a GPIb-lipid conjugateshowing superior retention property in blood can be prepared byintroducing polyalkylene oxide (hereinafter to be referred to as PAO)between GPIb and the lipid, and intensively studied further to completethe present invention.

[0007] Accordingly, the present invention relates to

[0008] (1) a conjugate comprising GPIb and a lipid that are bonded viaPAO (hereinafter to be also referred to as a GPIb-lipid conjugate),

[0009] (2) the conjugate of (1) above, wherein the GPIb is a GPIbitself, a GPIb fragment, a GPIb α chain or a GPIb α chain fragment,

[0010] (3) the conjugate of (1) above, wherein the GPIb is an analog, amutant, a modified compound, a derivative or a sugar chain adduct,having a von Willebrand Factor-binding capability almost of the samelevel as GPIb,

[0011] (4) the conjugate of (1) above, wherein the GPIb lacks atransmembrane site,

[0012] (5) the conjugate of (1) above wherein the lipid has a functionalgroup,

[0013] (6) the conjugate of (5) above, wherein the lipid having afunctional group is a phospholipid, a glycolipid, a fatty acid, aglyceride, a cholesterol or an amphipathic lipid,

[0014] (7) the conjugate of (5) above, wherein the functional group isan amino group, a carboxyl group, a thiol group or an aldehyde group,

[0015] (8) the conjugate of (1) above, wherein the PAO is polyethyleneglycol, polypropylene glycol, or glycol obtained by copolymerizingethylene oxide and propylene oxide,

[0016] (9) the conjugate of (1) above, wherein the PAO has a numberaverage molecular weight of 100-100000,

[0017] (10) the conjugate of (5) above, wherein the GPIb and the PAO,and the PAO and the lipid having a functional group are respectivelybonded chemically by a crosslinking agent,

[0018] (11) the conjugate of (5) above, wherein a molar ratio ofGPIb:PAO is 1:1-1:20 and a molar ratio of the PAO:lipid having afunctional group is 1:1,

[0019] (12) a complex comprising a GPIb-lipid conjugate and a free lipid(hereinafter to be also referred to as a GPIb lipid complex),

[0020] (13) the complex of (12) above, which is in the form of aliposome,

[0021] (14) the complex of (12) above, wherein the free lipid is aphospholipid, a glycolipid, a cholesterol, a fatty acid or a derivativethereof,

[0022] (15) the complex of (12) above, wherein a molar ratio of theGPIb:free lipid is 1:10-1:1000,

[0023] (16) the complex of (12) above, wherein the complex isaggregatable in the presence of ristocetin,

[0024] (17) the complex of (12) above, wherein the conjugate comprisingGPIb and the lipid bonded via PAO is prepared and then the complex ofsaid conjugate and a free lipid is prepared,

[0025] (18) a pharmaceutical composition containing the complex of (12)above,

[0026] (19) the pharmaceutical composition of (18) above, which is aplatelet substitute,

[0027] (20) the pharmaceutical composition of (18) above, which is anagent for the prophylaxis or treatment of a vascular disorder, avascular damage or thrombosis,

[0028] (21) a pharmaceutical agent for examination or diagnosis, whichcomprises, as an active ingredient, a labeling substance and the complexof (12) above,

[0029] (22) the pharmaceutical agent of (21) above, wherein the labelingsubstance is a radioisotope, a paramagnetic metal for MRI, an iodidecompound for X ray imaging, a fluorescent substance or a pigment,

[0030] (23) a drug-containing composition comprising a drug and thecomplex of (12) above as active ingredients, and

[0031] (24) the drug-containing composition of (23) above, wherein thedrug is a hemostatic agent, a vasoconstrictor, an antiinflammatoryagent, a thrombolytic agent, an anti-blood coagulator or ananti-platelet agent. The present invention is described in detail in thefollowing.

[0032] (I) Conjugate Consisting of GPIb-PAO-Lipid (GPIb-Lipid Conjugate)

[0033] The GPIb-lipid conjugate of the present invention is a conjugatewherein (A) GPIb and (B) a lipid are bonded via (C) PAO.

[0034] (A) GPIb

[0035] The GPIb to be used in the present invention may be any as longas it has a binding capability with vWF, and includes natural GPIbitself, and those obtained by subjecting one or plural amino acids ofthe amino acid sequence thereof to an optional mutation, such asdeletion, substitution, addition and modification, to the degree thatthe object of the present invention can be still achieved, that areexemplified by substitute, analog, mutant, modified compound,derivative, sugar chain adduct and the like of natural GPIb. Specificexamples include GPIb fragments such as α chain [His(1)-Leu(610)] ofGPIb, a fragment of a vWF binding region of the a chain (hereinafter tobe also referred to as a GPIb α chain fragment) and the like, GPIbfragments wherein transmembrane site has been deleted, and the like. Inthe present invention, GPIb fragments without the transmembrane site aremore preferably used.

[0036] More specific GPIb α chain fragments include His(1)-Cys(485),His(1)-Pro(340), His(1)-Thr(304), His(1)-Ala(302), His(1)-Arg(293)[JP-A-1-221394, EP-317278-A], Ala(165)-Leu(184), Gln(180)-Phe(199),His(195)-Leu(214), Asn(210)-Val(229), Glu(225)-Ala(244) andThr(240)-Tyr(259) [JP-A-1-100196], Asn(61)-Thr(75), Gln(71)-Ser(85),Thr(81)-Leu(95), Gln(97)-Arg(111), Leu(136)-Leu(150), Asn(210)-Ala(224),Gln(221)-Asp(235) and Ser(241)-Asp(255) [Japanese Patent Applicationunder PCT laid-open under kohyo No. 5-503708, WO91/09614] and the like.Examples of the substitute include a GPIb α chain fragment consisting ofHis(1)-Ala(302), wherein Gly(233) or Met(239) is substituted by Val, andthe like [WO93/16712]. These GPIb α chain fragments all lacktransmembrane site. The transmembrane site in the GPIb α chain isLeu(486)-Gly(514) (Proc. Natl. Acad. Sci. USA, Vol. 84, pp. 5615-5619,1987).

[0037] The GPIb can be prepared by any method and a method comprisingextraction and isolation from platelet membrane, a method using cellculture and a production method using genetic engineering areexemplified.

[0038] (B) Lipid

[0039] The lipid that is bonded to GPIb via PAO has a functional groupdirectly or indirectly bondable to PAO. Such functional group isexemplified by amino group (NH₂), carboxyl group (COOH), thiol group(SH), aldehyde group (CHO) and the like. As long as the functional groupcan form a direct or indirect bond with PAO, it is not limited to thoseexemplified.

[0040] The kind of the lipid is exemplified by phospholipid, glycolipid,fatty acid, glyceride, cholesterols and the like, with preference givento amphipathic ones.

[0041] The lipid is exemplified by phosphatidylethanolamine (hereinafterto be referred to as PE) and phosphatidylthioethanol (e.g.,1,2-dioleoyl-sn-glycero-3-phosphatidylthioethanol) for phospholipid. Asthe glycolipid, for example, ceramide, cerebroside, sphingosine,sulfatide, gangliosides and glyceroglycolipids (e.g.,galactosyldiacylglycerol) and the like are mentioned. The fatty acid isexemplified by saturated or unsaturated fatty acid having 12 to 18carbon atoms, which may be, for example, palmitic acid, oleic acid orlauric acid. As the glyceride, for example, monoglyceride, diglyceride,triglyceride and the like are mentioned. The cholesterols includecholesterol, cholesterol ester and allocholesterol. The functional groupin these lipids may be in the form of an acid halide, an acid anhydrideor an active ester, to enhance the reactivity with PAO and thecrosslinking agents to be mentioned later.

[0042] For an indirect bonding of a lipid and PAO, one obtained byreacting a crosslinking agent (i.e., spacer or linker) with thefunctional group of a lipid in advance can be also used. Examples ofsuch crosslinking agent include dicarboxylic acid, aminocarboxylic acid,bismaleimide compound, bishalocarbonyl compound, halocarbonylmaleimidecompound, dithiomaleimide, dithiocarboxylic acid, maleimidocarboxylicacid and the like. These crosslinking agents preferably have 2 to 10carbon atoms. These crosslinking agents and the functional group of alipid are reacted according to a conventional method.

[0043] The phospholipid formed by reacting said crosslinking agent withthe functional group is exemplified by PE-N-carbonyl amine (e.g.,PE-N-caproyl amine, PE-N-dodecanyl amine, PE-N-glutaryl amine and thelike), PE-N-carbonyI (e.g., PE-N-succinyl, PE-N-glutaryl (NGPE),PE-N-dodecanyl (NDPE) and the like), PE-N-dithioacylate (e.g.,PE-N-3-(2-pyridyldithio)-propionate), PE-N-maleimidoacylate (e.g.,PE-N-4-(p-maleimidophenyl)butyrate and the like), PE-N-biotinyl and thelike.

[0044] (C) PAO (Polyalkylene Oxide)

[0045] PAO is present between (A) GPIb and (B) a lipid and binds thetwo. The PAO is the same as polyalkylene glycol and exemplified bypolyethylene glycol (PEG), polypropylene glycol, glycol obtained bycopolymerization of ethylene oxide and propylene oxide, and the like.PAO has a high number average molecular weight of about 100-100,000,preferably about 1,000-10,000.

[0046] As the PAO, moreover, one obtained by reacting one or both of thetermini of PAO with a crosslinking agent (i.e., spacer, linker) inadvance can be also used. Examples of the crosslinking agent includedicarboxylic acid, aminocarboxylic acid, bismaleimide compound,bishalocarbonyl compound, halocarbonylmaleimide compound,dithiomaleimide, dithiocarboxylic acid, maleimidocarboxylic acid and thelike. These crosslinking agents and PAO are reacted according to aconventional method.

[0047] (D) Preparation of GPIb-Lipid Conjugate

[0048] The bond between GPIb and PAO, and the bond between PAO and alipid having a functional group are directly or indirectly formedchemically, i.e., via or not via a group derived from a crosslinkingagent. In the present invention, an indirect bond is preferred. Themolar mixing ratio of GPIb:PAO or a lipid having a functional group isabout 1:1-1:20, preferably about 1:1-1:10.

[0049] When the chemical bond between GPIb or a lipid having afunctional group and PAO cannot be formed directly, a method comprisingreacting the functional group of a lipid or PAO with the above-mentionedcrosslinking agent may be employed, or a known condensing agent, anactivating agent that activates the functional group in the lipid, adivalent crosslinking agent and the like may be used. Examples of thecondensing agent and the activating agent include carbodiimides (e.g.,N,N′-dicyclohexylcarbodiimide,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and the like),succinimides (e.g., N-hydroxysuccinimide, N-hydroxysulfosuccinimide(NHSS) and the like), a compound used for exchange reaction of thiolgroup (e.g., 5,5′-dithiobis(2-nitrobenzoic acid), 2,2′-dithiobispyridineand the like), and the like.

[0050] The divalent crosslinking agent may be a crosslinking agenthaving the same or different reactivity. Examples of the crosslinkingagent having the same reactivity include dimethyl adipimidate,disuccinimidyl suberate and the like, and examples of the crosslinkingagent having different reactivity includesuccinimidyl-3-(2-pyridyldithio)propionate,N-(6-maleimidocaproyloxy)succinimide,N-succinimidyl-6-maleimidohexanoate and the like.

[0051] As direct bond of GPIb or lipid and PAO, —COO— [e.g., obtained byester bonding of carboxyl group of GPIb or lipid (e.g., fatty acid) andhydroxyl group of PAO], —O— (e.g., obtained by ether bonding of hydroxylgroup of GPIb or lipid and hydroxyl group of PAO), —CONH— (e.g.,obtained by amide bonding of carboxyl group of GPIb or lipid and aminogroup introduced into PAO), —CH═N— (e.g., obtained by Schiff bonding ofaldehyde group on sugar chain of GPIb or aldehyde group introduced intolipid and amino group introduced into PAO), —CH₂NH— (obtained by furtherreduction of Schiff bond), —NH— and the like are mentioned.

[0052] Of the specific methods for indirectly bonding GPIb or lipid andPAO, the mode of bonding of the crosslinking agent (inclusive ofdivalent crosslinking agent, hereinafter the same) and PAO isexemplified by —COO—, —O—, —CONH—, —CH═N—, —CH₂NH—, —NH— and the like,like those mentioned above. As the mode of bonding of GPIb or lipid andthe crosslinking agent, there are mentioned, besides those similar tothe above-mentioned, —S—S— [obtained by disulfide bonding of thiol groupof GPIb or lipid (e.g., phosphatidylthioethanol) and dithio moiety ofcrosslinking agent such as dithiomaleimide, dithiocarbonyl compound andthe like], —S— (obtained by bonding thiol group of GPIb or lipid andcrosslinking agent such as maleimide compound, halocarbonyl compound andthe like, by reductive alkylation), —NH— (e.g., obtained by reactingamino group of GPIb or lipid (e.g., PE) and maleimide moiety ofcrosslinking agent, such as maleimidocarboxylic acid and the like), —Co—and the like.

[0053] The GPIb-lipid conjugate may be prepared in the presence of asurfactant. The surfactant is not particularly limited as long as itsolubilizes lipid. The use of a nonionic surfactant is preferable, sothat the structure of GPIb will not be influenced. In particular, anonionic surfactant having a high critical micelle concentration (CMC),such as a CMC of not less than 1 mM, is preferable. Examples thereofinclude octyl glucoside (e.g., n-octyl-β-D-glucoside), octylthioglucoside (e.g., n-octyl-β-D-thioglucoside),3-[(3-cholamidopropyl)-dimethylammonio]propanesulfate (CHAPS) andN,N-bis(3-D-gluconamidopropyl)deoxycholamide (deoxy-BIGCHAP). The mixingratio of lipid:surfactant is preferably about 0.01:1-0.1:1 by mole.

[0054] (E) Activated Lipid Consisting of Reactive Substituent-PAO-Lipid(Hereinafter to be Also Referred to Simply as an Activated Lipid)

[0055] As an intermediate for synthesizing a conjugate (GPIb-lipidconjugate) consisting of GPIb-PAO-lipid, an activated lipid consistingof reactive substituent-PAO-lipid can be used. As used herein, by the“activated lipid” is meant a bonded product of PAO and lipid having areactive substituent on the terminal of PAO. PAO and the lipid aredirectly or indirectly bonded chemically via or not via a group derivedfrom a crosslinking agent. The reactive substituent is subject to noparticular limitation as long as it has an action of activating a bondwith a protein. For example, it can be obtained by reacting succinimide,maleimide and the like with the terminal of PAO. The mode of the bondbetween the reactive substituent and PAO, and PAO and a lipid in theactivated lipid is as shown under the above-mentioned (D) Preparation ofGPIb-lipid conjugate.

[0056] By directly or indirectly bonding activated lipid and GPIbchemically, i.e., via or not via a crosslinking agent, a GPIb-lipidconjugate can be obtained.

[0057] As the activated lipid, the following compounds are specificallyexemplified. These activated lipids are known and can be producedaccording to a known method. Alternatively, a commercially availableproducts can be used.

[0058] DSPE-34HCSI:distearoylphosphatidyl-N-(succinimidylsuccinyl-polyoxyethylene-succinyl)ethanolamine.The number average molecular weight of PEG is 3400.

[0059] DSPE-2OHMAL:distearoylphosphatidyl-N-(maleimidopropionylamino-polyoxyethylene-oxycarbonyl)-ethanolamine.The number average molecular weight of PEG is 2000.

[0060] (F) Property of GPIb-Lipid Conjugate

[0061] The GPIb-lipid conjugate of the present invention comprises GPIband PAO or lipid in a constituting molar ratio of about 1:1-1:20. Theconstituting molar ratio of PAO and lipid is 1:1.

[0062] (II) Complex of GPIb-Lipid Conjugate and Free Lipid (GPIb LipidComplex)

[0063] The complex of the present invention containing theabove-mentioned GPIb-lipid conjugate and free lipid can take the form ofa liposome, micell or lipid emulsion, with preference given to aliposome. As an example of the complex of the present invention, a GPIblipid complex in the form of a liposome is explained. The explanation ofthe following GPIb lipid complex applies to other forms besidesliposome.

[0064] (A) Free Lipid

[0065] The lipid to be used here is free of limitation as long as it cantake the form of a lipid complex, such as a liposome, and may be usedalone or in combination with other lipid. Examples of such lipid includephospholipid, glycolipid, cholesterol, fatty acid and derivativesthereof. The free lipid to be used in the present invention for formingthe complex may be any non-toxic lipid as long as it is physiologicallyacceptable and metabolizable. Note that a free lipid means a lipid otherthan a lipid constituting the GPIb-lipid conjugate, and may behomologous or heterologous with the lipid constituting the GPIb-lipidconjugate.

[0066] Examples of the phospholipid include phosphatidylcholine(hereinafter to be referred to as PC), phosphatidylserine, phosphatidicacid, phosphatidylglycerol, phosphatidylinositol, sphingomyelin, dicetylphosphate, cardiolipin, lysophosphatidylcholine and the like. Theselipids may be extracted and purified from a natural material such assoybean oil or egg yolk, or prepared by hydrogenation thereof tosaturate the constituent fatty acid (hydrogenated phospholipid), orobtained by substituting the constituent fatty acid with specific fattyacid, such as palmitic acid and myristic acid (e.g.,diacylphosphatidylcholine, diacylphosphatidyl glycerol and the like).Specific examples thereof include purified egg yolk lecithin,hydrogenated purified soybean lecithin, egg yolk-originatedphosphatidylcholine, dipalmitoylphosphatidylcholine,dipalmitoylphosphatidyl glycerol, distearylphosphatidylcholine,dimyristylphosphatidylcholine and the like.

[0067] Glycolipid may be, for example, ceramide, cerebroside,sphingosine, sulfatide, gangliosides or glyceroglycolipids.

[0068] The cholesterols include cholesterol, cholesterol ester andallocholesterol.

[0069] The fatty acid is exemplified by oleic acid, lauric acid,myristic acid, palmitic acid and stearic acid. Examples of the lipidderivative include polyoxyethylene derivative having phosphatidylethanolamine and fatty acid, and polysaccharide derivative having fattyacid and cholesterol. Specifically, it may bedistearyl-N-(monomethoxypolyethylene glycol succinyl)-phosphatidylethanolamine, polyoxyethylene palmitate,N-[2-(stearoylcarboxy-amino)ethyl]carbamoyl methyl mannan orN-[2-(cholesterylcarboxy-amino)ethyl]carbamoylmethylpluran.

[0070] (B) Formation of Complex

[0071] As the mixing ratio of GPIb-lipid conjugate:free lipid, the molarmixing ratio of GPIb:free lipid is about 1:10-1:1000, preferably about1:50-1:200.

[0072] A GPIb lipid complex (e.g., liposome) can be prepared by, forexample, surfactant removal method, hydrate method, ultrasonication,reversed phase evaporation method, freeze-thaw method, ethanol injectionmethod, extrusion method or high pressure emulsification. The surfactantremoval method is generally gel filtration, dialysis or ultrafiltration.In the present invention, a method comprising forming a complex afterbonding GPIb and a lipid via PAO, or forming a lipid complex afterpreparation of a GPIb-lipid conjugate, is more preferable. It is alsopossible to prepare a GPIb lipid complex by adding GPIb afterpreparation of a lipid complex (e.g., liposome) consisting of the lipidof (I)(B) and the free lipid of (II)(A), thereby to bond the lipid of(I)(B) and GPIb via PAO. The GPIb lipid complex can be isolated andpurified by a method known per se, such as centrifugation and gelfiltration.

[0073] The production method of the GPIb lipid complex is exemplified inthe following.

[0074] (i) Method for Forming a GPIb Lipid Complex After Preparation ofa GPIb-Lipid Conjugate

[0075] A lipid [(I)(B)] is solubilized with a surfactant and bonded withPAO to give a bonded product. This bonded product, GPIb and a free lipid[(II)(A)] are mixed in a suitable aqueous solvent to allow bonding ofGPIb and the lipid [(I)(B)] via PAO, and then the surfactant is removedto form a GPIb lipid complex. Alternatively, a bonded product of thelipid [(I)(B)] and PAO and GPIb are mixed in the presence of asurfactant to prepare a GPIb-lipid conjugate, a free lipid [(II)(A)] ismixed and the surfactant is removed, thereby to give a GPIb lipidcomplex. The unreacted GPIb, lipid [(I)(B)], free lipid [(II)(A)] andthe like may be separated and removed to give a purified product. Thesurfactant is the same as those mentioned above. The mixing ratio (freelipid [(II)(A)]:surfactant) is about 0.001:1-0.1:1 (molar ratio).

[0076] (ii) Method for Bonding GPIb After Preparation of a Lipid Complex(e.g., Liposome) to Give GPIb Lipid Complex

[0077] The bonded product of lipid [(I)(B)] and PAO, and free lipid[(II)(A)] are dissolved and mixed in an organic solvent such aschloroform and ethanol, and the organic solvent is removed to give athin lipid membrane. A suitable aqueous solvent is added and the mixtureis treated by a known method, such as shaking and stirring, to give alipid complex. GPIb is added to form a bond of GPIb and the lipid viaPAO, whereby a GPIb lipid complex is formed. The unreacted GPIb, lipidand the like may be separated and removed to give a purified product.

[0078] For this method, a free lipid [(II)(A)] that does not react/bindwith GPIb is used. Specially, phosphatidylcholine,lysophosphatidylcholine and the like are used.

[0079] The proportion of GPIb to free lipid [(II)(A)] in the obtainedGPIb lipid complex is 0.01-10 parts by weight, preferably 0.1-5 parts byweight, per part by weight of the free lipid [(II)(A)].

[0080] The obtained GPIb lipid complex has a particle size of about50-500 nm, preferably about 100-400 nm. The number of GPIb molecules perparticle is 100-10000, preferably 250-3000 and the surface density ofGPIb is 10¹⁰-10¹³, preferably 10¹¹-10¹².

[0081] The GPIb lipid complex (e.g., liposome) has a structure ofmultilamella vesicle (MLV), small unilamella vesicle, large unilamellavesicle and the like. It may be coated with a hydrophilic polymer suchas polyethylene glycol (PEG), pluronic (registered trademark)(polyoxyethylene polyoxypropylene block copolymer) and the like.

[0082] Where necessary, the obtained GPIb lipid complex is washed with aphysiologically acceptable aqueous solution, sterilized by filtration,dispensed and formulated into a liquid, pellet or suspensionpreparation.

[0083] The complex can be processed by a method known to be usable forthe preparation of pharmaceutical products. The above-mentionedpreparations may be provided as lyophilized preparations upon freezing aliquid preparation and drying same under reduced pressure. Forlyophilization, monosaccharides (e.g., glucose), disaccharides (e.g.,sucrose) and the like may be added.

[0084] The preparations of GPIb lipid complex may contain, as astabilizer, a polymer selected from albumin, dextran, vinyl polymer,gelatin and hydroxylethyl starch.

[0085] The polymer may be incorporated into the gaps present in saidlipid complex together with a drug. Alternatively, the polymer may beadded to or contained in said lipid complex preparation containing adrug. This means that the polymer may be added or incorporated outsidethe liposome. It is needless to say that it can be incorporated bothinside and outside the lipid complex.

[0086] The stabilizer is added in an amount of 0.5-10 parts by weight,preferably 1-5 parts by weight, per part by weight of the free lipid[(II)(A)].

[0087] (III) Use of GPIb Lipid Complex as Pharmaceutical

[0088] The GPIb lipid complex of the present invention can be embodiedas a diagnostic of the location of vascular lesion or thrombusformation, von Willebrand Factor deficiency and the like by adding alabeling substance (so-called a marker). Examples of such labelingsubstance include RI (radioisotope), paramagnetic metal for MRI, iodidecompound for X ray imaging, fluorescent substance and pigment.

[0089] RI is exemplified by ³H, ¹⁴C, ^(99m)Tc, ¹²³I, ¹²⁵I, ¹³¹I,^(87m)Sr, ^(113m)In, ¹⁹⁷Hg and the like. The paramagnetic metal for MRIis exemplified by divalent ion and trivalent ion of a paramagnetic metalof chromium (Cr), gadrinium (Gd), manganese (Mn), iron (Fe), cobalt(Co), nickel (Ni), praseodymium (Pr), neodium (Nd), samarium (Sm),ytterbium (Yb), terbium (Tb), dysprosium (Dy), hormium (Ho), erbium(Er), copper (Cu) and the like, with preference given to divalent ionand trivalent ion of gadrinium, terbium, dysprosium, hormium, erbium andiron.

[0090] The iodide compound for X ray imaging may be a known compound forX ray imaging. Examples thereof include adipiodone, amidotrizoic acid,iotalamic acid, iopanoic acid, iobenzamic acid, iopodate, tyropanoicacid, iopydol, iopydone, propyliodone, iodamide and salts thereof (e.g.,sodium salt and meglumine salt). The fluorescent substance may be, forexample, fluorescein isothiocyanate (FITC), carboxyfluorescein (CF) andthe like.

[0091] These labeling substances can be encapsulated in a lipid complex(e.g., liposome) by a known method. For example, it can be enclosed in alipid complex in the form of a salt or after chelating with a chelatingagent such as ethylenediaminetetraacetic acid (EDTA) anddiethylenetriaminepentaacetic acid (DTPA). In the case of ^(99m)Tc, forexample, sodium pertechnetate, technetium polyphosphate or ^(99m)Tc DTPAcan be used.

[0092] The pharmaceutical agent for examination or diagnosis, whichcontains a labeling substance and the GPIb lipid complex of the presentinvention may be a GPIb lipid complex of the present invention, which isprepared according to a method mentioned above and using GPIb labeledwith a labeling substance (e.g., RI).

[0093] The GPIb lipid complex of the present invention is cumulative atthe vascular lesion and thus, may be formed into a compositioncontaining a drug (drug vehicle). The drug to be contained is free ofany particular limitation as long as it proves physiologically andpharmacologically effective upon accumulation thereof at the vascularlesion, and may be, for example, hemostatic agent, vasoconstrictor,antiinflammatory agent, thrombolytic agent, anti-blood coagulator,anti-platelet agent and the like.

[0094] The hemostatic agent is exemplified by carbazochrome, bloodcoagulation factor (FVIII, FIX), thrombin, antiplasmin agent (e.g.,ε-aminocaproic acid and tranexamic acid), protamine sulfate, etamsylate,phytonadione and conjugated estrogen (e.g., sodium estrone sulfate andsodium equilin sulfate) and the like.

[0095] The vasoconstrictor is exemplified by noradrenaline,norfenefrine, phenylephrine, metaraminol, methoxamine, prostaglandin F₁α, prostaglandin F₂ α, thromboxane A₂ and the like. The antiinflammatoryagent is exemplified by steroidal antiinflammatory agent (e.g.,dexamethasone, hydrocortisone, prednisolone, betamethasone,triamcinolone, methylprednisolone and the like), non-steroidalantiinflammatory agent (e.g., indometacin, acemetacin, flurbiprofen,aspirin, ibuprofen, flufenamic acid, ketoprofen, and the like) and thelike.

[0096] The thrombolytic agent is exemplified by plasmin, tissueplasminogen activator, urokinase, precursors thereof and derivativesthereof. The anti-blood coagulator is exemplified by acidicmucopolysaccharide (e.g., heparin), coumarin anti-blood coagulator,natural extract (e.g., hirudine) and derivatives thereof,physiologically active substances (e.g., thrombomodulin, active proteinC, and the like) and the like. The anti-platelet agent is exemplified byaspirin, ticlopidine, cilostazol, prostacyclin and the like. These drugscan be encapsulated in a lipid complex by a known method.

[0097] The inventive GPIb lipid complex is administered in an amount ofabout 0.001-1000mg as GPIb per day. The dose can be appropriatelychanged depending on the sex, age and symptoms of patients.

[0098] The GPIb lipid complex is more preferably administeredparenterally. To be specific, it is administered by intravascular(intraarterial or intravenous) injection, intravenous drip, subcutaneousadministration, local administration, intramuscular administration andthe like.

[0099] The pharmaceutical composition containing the inventive GPIblipid complex is useful as a platelet substitute, a pharmaceuticalproduct for the prophylaxis or treatment of angiopathy, vascular damagesand thrombosis, a diagnostic for vWF deficiency and the like, abiological or medical reagent, a reagent for screening plateletaggregation suppressant or antithrombosis, and the like. It is alsouseful for a diagnostic for finding the location of vascular lesion orthrombus formation, or a therapeutic agent thereof.

EXAMPLES

[0100] The present invention is explained in more detail in thefollowing by way of Examples and Experimental Examples, which are not tobe construed as limitative. The GPIb subjected to the Examples andExperimental Examples of the present invention was a GPIb α chainfragment [His(1)-Arg(293), molecular weight 45000, JP-A-9-208599]produced by genetic engineering using a CHO cell.

Example 1

[0101] To physiological saline (100 μL) containing GPIb (100 mg/mL) wereadded egg yolk phosphatidylcholine (EPC) (221 mg), cholesterol (45.4 mg)and octyl glucoside (OG) (2.33 g), dissolved in water for injection (1.5mL). The amount of EPC corresponded to a 100-fold molar ratio of GPIb.Then, DSPE-34HCSI (purchased from NOF Corporation) dissolved in HEPESbuffer (pH 8.0) containing 0.1% OG was added. The amount of DSPE-34HCSIadded was a 10-fold molar amount of GPIb. After reaction at 4° C. for 16hr, OG was removed by gel filtration (carrier was Sephadex G-75,Pharmacia Biotech) to give a liposome. GPIb liposome was recovered bycentrifugal separation by CsCl density gradient method. The conditionstherefor were as follows. CsCl was dissolved in a sample (1-1.5 mL) to aconcentration of 35%, and 0.5 mL of 25% CsCl and 0.1 mL of physiologicalsaline were layered thereon and treated at 214000×g for 10 min. Theliposome-containing fraction was recovered and dialyzed overnightagainst a dialysis membrane (molecular weight of fraction 10,000 dalton,Slide-A-Lyzer 10K, Pierce Biotechnology, Inc.) using physiologicalsaline. The prepared GPIb liposome had a PC concentration of 0.61 mg/mL,a GPIb concentration of 0.24 mg/mL, and an average particle size of 0.25μm.

Example 2

[0102] To 1,3,4,6-tetrachloro-3α,6α-diphenylglycouril (200 μg) wereadded Na¹²⁵I solution (448 MBq/mL, 30 μL), and then physiological saline(100 μL) containing GPIb (100 mg/mL), and the mixture was reacted atroom temperature for 10 min. 25 mM NaI (100 μL) was added and themixture was subjected to gel filtration (MicroSpin G-25, PharmaciaBiotech) and ultrafiltration (Ultrafree-MC10K, Millipore Corporation) togive iodine-labeled GPIb (¹²⁵I-GPIb).

[0103] The operation followed that of Example 1 except theabove-mentioned ¹²⁵I-GPIb was used instead of GPIb. The prepared GPIbliposome had a PC concentration of 3.54 mg/mL, a GPIb concentration of1.00 mg/mL, an average particle size of 0.33 μm, and radioactivity of9.3×10⁷ cpm/mL.

Experimental Example 1

[0104] The aggregation capability of the GPIb liposome was confirmed.The GPIb liposome prepared in Example 1 was adjusted to a PCconcentration of 0.2 mg/mL. To the solution (200 μL) was added 2 mg/mLof a vWF solution (25 μL) and the mixture was incubated and stirred at37° C. After 2 min, 15 mg/mL of a ristocetin solution (25 μL) was mixed.The. aggregate was measured for the total of 15 min from immediatelybefore incubation using an aggregometer (AG-10, Kowa Company, Ltd.). Thetotal of the values of aggregate number multiplied by scatteringintensity was calculated and used as an index of the degree of aggregateformation. The results are shown in Table 1.

[0105] A similar experiment was performed using, as a control, a GPIbliposome (described in Example 1 of JP-A-9-208599) without PEG. Theresults are shown in Table 2. TABLE 1 Degree of aggregate formation(×10⁴) Small Medium Large Test time aggregate aggregate aggregateImmediately before 0 0 0  5 min later 3.1 4.0 9.5 10 min later 2.0 2.810.3 15 min later 1.3 1.3 8.0

[0106] TABLE 2 Degree of aggregate formation (×10⁴) Small Medium LargeTest time aggregate aggregate aggregate Immediately before 0 0 0  5 minlater 0.4 0.2 0 10 min later 1.7 1.7 3.0 15 min later 1.8 2.0 7.8

[0107] The GPIb liposome of the present invention specifically reactedwith vWF in the presence of ristocetin and formed an aggregate. This hasdemonstrated that the GPIb liposome is useful as a platelet substitute.In addition, the GPIb liposome of the present invention has beenclarified to have improved the aggregation capability as compared to thecontrol.

Experimental Example 2

[0108] The hemodynamics of the GPIb liposome was confirmed. The iodinelabeled GPIb liposome prepared in Example 2 was administered to Hartleyguinea pigs (n=3) at the dose of 1 mg/kg body weight in GPIb into thefemoral vein. The blood was drawn from the orbital with the lapse oftime for 60 min after the administration and the radioactivity of blood(100 μL) was counted on a gamma counter (1 min). A similar experimentwas performed using, as a control, a GPIb liposome (described in Example1 of JP-A-9-208599) without PEG. The results are shown in Table 3. TABLE3 blood concentration Time after (%: relative to dose) administrationExample 2 control  2 min later 53 18  5 min later 27 3 10 min later 16 230 min later 9 6 60 min later 8 7

[0109] The GPIb liposome of the present invention has been clarified tohave improved the retention property in blood as compared to thecontrol.

Industrial Applicability

[0110] The GPIb lipid complex of the present invention can bind with vWFand form an aggregate. Thus, it is expected to have a potential forpractical use in a wide range as a platelet substitute, a pharmaceuticalagent for the prophylaxis or treatment of angiopathy, vascular damageand thrombosis, a diagnostic for vWF deficiency and the like, abiological or medical reagent, a reagent for screening plateletaggregation suppressant or antithrombosis, and the like. The GPIb lipidcomplex of the present invention is also useful as a diagnostic forfinding the location of vascular lesion or thrombus formation, or atherapeutic agent thereof, since it specifically accumulates at vascularlesions. Moreover, the GPIb lipid complex of the present invention isalso superior in retention property in blood, which enables continuousexpression of a pharmacological action.

[0111] The GPIb-lipid conjugate of the present invention is highlyutilizable as an active ingredient of the GPIb lipid complex of thepresent invention.

[0112] This application is based on application No. 2000-57449 filed inJapan, the contents of which are incorporated hereinto by reference.

What is claimed is
 1. A conjugate comprising GPIb and a lipid that arebonded via polyalkylene oxide.
 2. The conjugate of claim 1, wherein theGPIb is a GPIb itself, a GPIb fragment, a GPIb α chain or a GPIb α chainfragment.
 3. The conjugate of claim 1, wherein the GPIb is an analog, amutant, a modified compound, a derivative or a sugar chain adduct,having a von Willebrand Factor-binding capability almost of the samelevel as GPIb.
 4. The conjugate of claim 1, wherein the GPIb lacks atransmembrane site.
 5. The conjugate of claim 1 wherein the lipid has afunctional group.
 6. The conjugate of claim 5, wherein the lipid havinga functional group is a phospholipid, a glycolipid, a fatty acid, aglyceride, a cholesterol or an amphipathic lipid.
 7. The conjugate ofclaim 5, wherein the functional group is an amino group, a carboxylgroup, a thiol group or an aldehyde group.
 8. The conjugate of claim 1,wherein the PAO is polyethylene glycol, polypropylene glycol, or glycolobtained by copolymerizing ethylene oxide and propylene oxide.
 9. Theconjugate of claim 1, wherein the PAO has a number average molecularweight of 100-100000.
 10. The conjugate of claim 5, wherein the GPIb andthe PAO, and the PAO and the lipid having a functional group arerespectively bonded chemically by a crosslinking agent.
 11. Theconjugate of claim 5, wherein a molar ratio of GPIb:PAO is 1:1-1:20 anda molar ratio of the PAO:lipid having a functional group is 1:1.
 12. Acomplex comprising the conjugate of claim 1 and a free lipid.
 13. Thecomplex of claim 12, which is in the form of a liposome.
 14. The complexof claim 12, wherein the free lipid is a phospholipid, a glycolipid, acholesterol, a fatty acid or a derivative thereof.
 15. The complex ofclaim 12, wherein a molar ratio of the GPIb:free lipid is 1:10-1:1000.16. The complex of claim 12, wherein the complex is aggregatable in thepresence of ristocetin.
 17. The complex of claim 12, wherein theconjugate comprising GPIb and the lipid bonded via PAO is prepared andthen a complex of said conjugate and a free lipid is prepared.
 18. Apharmaceutical composition comprising the complex of claim
 12. 19. Thepharmaceutical composition of claim 18, which is a platelet substitute.20. The pharmaceutical composition of claim 18, which is an agent forthe prophylaxis or treatment of a vascular disorder, a vascular damageor thrombosis.
 21. A pharmaceutical agent for examination or diagnosis,which comprises, as an active ingredient, a labeling substance and thecomplex of claim
 12. 22. The pharmaceutical agent of claim 21, whereinthe labeling substance is a radioisotope, a paramagnetic metal for MRI,an iodide compound for X ray imaging, a fluorescent substance or apigment.
 23. A drug-containing composition comprising a drug and thecomplex of claim 12 as active ingredients.
 24. The drug-containingcomposition of claim 23, wherein the drug is a hemostatic agent, avasoconstrictor, an antiinflammatory agent, a thrombolytic agent, ananti-blood coagulator or an anti-platelet agent.